WO2002066673A1 - Technique de preparation de levure destinee a un essai de fermentation - Google Patents

Technique de preparation de levure destinee a un essai de fermentation Download PDF

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Publication number
WO2002066673A1
WO2002066673A1 PCT/JP2002/001365 JP0201365W WO02066673A1 WO 2002066673 A1 WO2002066673 A1 WO 2002066673A1 JP 0201365 W JP0201365 W JP 0201365W WO 02066673 A1 WO02066673 A1 WO 02066673A1
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WO
WIPO (PCT)
Prior art keywords
yeast
weight
malt
medium
fermentation test
Prior art date
Application number
PCT/JP2002/001365
Other languages
English (en)
Japanese (ja)
Inventor
Nobuyuki Hayashi
Original Assignee
Kirin Beer Kabushiki Kaisha
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kirin Beer Kabushiki Kaisha filed Critical Kirin Beer Kabushiki Kaisha
Priority to DE60210749T priority Critical patent/DE60210749T2/de
Priority to AU2002232224A priority patent/AU2002232224B2/en
Priority to JP2002566377A priority patent/JP3916567B2/ja
Priority to EP02712420A priority patent/EP1362923B1/fr
Priority to US10/468,295 priority patent/US7713717B2/en
Priority to CA002438668A priority patent/CA2438668A1/fr
Publication of WO2002066673A1 publication Critical patent/WO2002066673A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts

Definitions

  • the present invention relates to a method for preparing an yeast used for testing the quality of malt used for beer brewing, and a medium used for the method for preparing such a yeast.
  • Malt used in beer brewing is a raw material that has a great influence on determining the taste and aroma of beer, and is said to be the life of beer.
  • Malt contains various components such as protein, starch, and extract, and the quality of the malt may be determined by the composition of such components.However, the fermentability of wort using such malt is evaluated. It is considered the most important factor in determining malt quality.
  • a beer is usually produced by grinding wort, saccharifying starch, filtering, and boiling to produce wort, and then adding yeast to the cooled wort and fermenting it. If the malt quality is good, yeast fermentation will be vigorous and beer with excellent flavor can be produced, but if the malt quality is poor, fermentation will not proceed slowly and products with low commercial value will be produced. May be manufactured.
  • malt also contains a substance that promotes yeast flocculation, and some barley crops in that year contain a large amount of substances that promote yeast flocculation. It can be lost.
  • a substance that promotes such aggregation is generally called a precoagulation factor, and it is important in fermentation management to know how much the precoagulation factor is contained in malt.
  • the grinding scale of the disc mill was set so that the distance between the discs was exactly 1.0 mm, and 300 g of malt was disc-milled to a total volume of 2 L.
  • a saccharification beaker stainless steel beaker.
  • Add 1.8 L of tap water at 46-47 ° C to a saccharification beaker containing the crushed malt place the mixture in a large saccharification tank with the water temperature adjusted to 45 ° C, and continue stirring while the temperature of the congress method (4 Saccharify at 5 ° C for 30 minutes ⁇ 1 ° C / min ⁇ 70 ° (: 1 hour) Measure the filtration speed of mashed moromi using a conical filter cloth.
  • the wort containing yeast thus obtained was transferred to a K1 fermentation tube (500 m1 volume, diameter 25 mm, height 125 mm), and kept at 8 ° C soil and 0.2 ° C for 7 days. Place.
  • Control malt is used as a control.
  • the malt evaluation fermentation test described above which has been established as a standard analytical method for evaluating the fermentation characteristics of malt, is an excellent method.However, during the examination process to obtain more reproducible test results, the yeast We have learned that physiological condition may be a factor affecting the test results.
  • the malt evaluation and fermentation test is a kind of bioassay using yeast, and the yeast (factory yeast) collected after actually using it in a beer factory is usually used for the malt evaluation and fermentation test described above.
  • An object of the present invention is to provide a method for preparing yeast for use, which can obtain more reproducible test results in a malt evaluation and fermentation test at a laboratory level without being affected by the operation conditions of a factory and the like. To provide a culture medium. Disclosure of the invention
  • the present inventors have conducted intensive research to solve the above-mentioned problems, and in a malt evaluation and fermentation test at a laboratory level, control the growth of yeast using an appearance extract as an index, and always evaluate yeast having the same physiological state as malt evaluation and fermentation. More reproducible when a liquid medium containing 6 to 12% by weight of sugar that yeast can ferment and a total amount of free amino acids of 140 to 300 mg ZL is used as a medium for the test. To complete the present invention. I came to.
  • the present invention uses a liquid medium containing 6 to 12% by weight of sugar that can be fermented by yeast and a total amount of free amino acid of 1 AOOSOO OmgZL as a blending component, until the sugar content reaches 3.0 ° P or less.
  • a method for preparing a yeast for malt evaluation and fermentation test, wherein the yeast is recovered after stirring culture of the yeast (claim 1), a sugar capable of fermenting the yeast at 6 to 12% by weight, and a total amount of free amino acid of 1 Using a liquid medium containing 400 to 300 mg / L, perform culturing with stirring (primary culturing) at 10 to 20 ° C for 2 to 4 days.
  • a method for preparing a yeast for malt evaluation and fermentation test wherein the yeast is recovered after leaving it in a room (Claim 2), and a sugar capable of fermenting the yeast of 6 to 12% by weight.
  • a liquid medium containing the total amount of free amino acids 140 to 300 mg ZL glucose 0 to 2.5% by weight, maltose 6.0 to 9.0% by weight, sorbitol 0 to 2.5% 3.
  • the yeast for malt evaluation and fermentation test according to claim 1 or 2 wherein a liquid culture medium containing 0.3% to 1.0% by weight of peptone, 0.3% to 1.0% by weight of peptone, and 0.3% to 1.0% by weight of yeast is used. And a liquid medium containing 6 to 12% by weight of sugar that can be fermented by yeast and a total amount of free amino acids of 1400 to 300 mg of ZL, 1.5% by weight of glucose and maltose.
  • the malt evaluation and fermentation test according to claim 3 wherein a liquid medium containing 6.5% by weight, sorbitol 2.0% by weight, peptone 0.2% by weight, and yeast extract 0.4% by weight is used.
  • the present invention also provides glucose 0-2.5% by weight, maltose 6.0-9.0% by weight, sorbitol 0-2.5% by weight, peptone 0-1.0% by weight, yeast extract 0 Malt evaluation characterized by containing 3 to 1.0% by weight.
  • a yeast preparation medium (claim 6) in a fermentation test, glucose 1.5% by weight, maltose 6.5% by weight, sorbitol.
  • FIG. 1 is a diagram showing the results of measurement of the progress of the tertiary culture by the ⁇ D value (OD 800 nm) at a wavelength of 800 nm.
  • Fig. 2 shows the results of the malt evaluation and fermentation test of the present invention, using control malt and defective malt, for factory yeast, yeast prepared using O medium, and yeast prepared using HB medium. It is.
  • FIG. 3 is a diagram showing the results obtained by repeating the malt evaluation fermentation test of the present invention four times using factory yeast and yeast prepared using HB medium.
  • a liquid containing 6 to 12% by weight of sugar that can be fermented by yeast and a total amount of 140 to 300 mg of free amino acid as a compound component The yeast is stirred and cultivated in a medium until the sugar content becomes 3.0 ° P or less, and then the yeast is recovered.
  • a method for recovering the yeast, 6 to 12% by weight of sugar that the yeast can ferment, and a total amount of free amino acids of 140 Using a liquid medium containing 0 to 300 mg / L, perform agitating culture (primary culture) at 10 to 20 ° C for 2 to 4 days.
  • the method for recovering the precipitated yeast is not particularly limited, and the malt evaluation and fermentation test described above is, for example, reported by the present applicant (EUROPEAN BREWERY CONVENTION MONOGRAPH XXIII EBC -SYMPOSIUM MALTING TECHNOLOGY ANDERNACHGERMANY NOVEMBER 1994 , 110-136) can be used.
  • yeast streaked on a normal malt agar slant medium can be used, and the streaked yeast cultured should be stored at 4 ° C for about half a year. Can be.
  • a tertiary culture of yeast amount 1. 0 X 1 0 1 Q ⁇ 1. 2 X 1 0 10 ce 1 2 primary cultures such that I s Is preferably added to 4 L of a liquid medium.
  • sugars that can be fermented by the yeast include Darcos, maltose, sucrose, fructose, and the like, and those containing glucose and maltose are preferable. 12% by weight, preferably 8 to 12% by weight is added.
  • the free amino acid containing the total amount of 140 to 300 mgOgZL include Peptone yeast extract, but are not limited thereto. The total amount of free amino acids can be determined by an ordinary method using an amino acid analyzer.
  • the total amount of free amino acids means free aspartic acid, threonine, serine, glutamic acid, proline (imino acid), glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, fenylalanine, tryptophan, lysine, histidine.
  • sugar that yeast can ferment 6 As a liquid medium containing 1 to 12% by weight and the total amount of free amino acids 1400 to 300mg / L, glucose 0 to 2.5% by weight, maltose 6.0 to 9.0% by weight, A liquid medium containing sorbitol 0-2.5% by weight, peptone 0-: L.
  • yeast extract 0.3-1.0% by weight is preferable, and glucose 1.5% by weight
  • a liquid medium (HB medium) containing 6.5% by weight of sorbitol, 2.0% by weight of sorbitol, 0.2% by weight of peptone, and 0.4% by weight of yeast extract is particularly preferred.
  • the culture medium used in the examination of the culture conditions includes YM medium generally used for culturing yeast (Dulcose 1%, peptone 0.5%, malt extract 0.3%, yeast extract 0.3%), etc. 0 medium [Dalcose 2.5%, Maltose 7.5%, Peptone 0.5%, Yeast extract 0.3%; pH 6. 2] was used.
  • YM medium generally used for culturing yeast (Dulcose 1%, peptone 0.5%, malt extract 0.3%, yeast extract 0.3%), etc.
  • 0 medium [Dalcose 2.5%, Maltose 7.5%, Peptone 0.5%, Yeast extract 0.3%; pH 6. 2] was used.
  • One platinum loop of yeast cultivated on a malt agar medium was inoculated into 8 ml of the above-described O medium in the tester, and cultured with stirring at 14 ° C for 2 to 4 days (primary culture).
  • the entire amount of the primary culture was added to 150 ml of the 0-medium in a 300 ml Erlenmeyer flask, and cultured with stirring at 14 ° C. for 3 to 4 days (secondary culture).
  • Secondary culture About 1.
  • l Xl O i Q celi S obtained from the secondary culture was added to 4 L of the O medium in a 5 L Duran bottle, and the mixture was stirred at 9 ° C for 7 to 9 days. (Tertiary culture) with stirring at 250 rpm.
  • FIG. 1 shows the results of measurement of the progress of the tertiary culture by the OD value ( ⁇ D 800 nm) at a wavelength of 800 nm.
  • OD absorbance
  • the absorbance differs depending on the manufacturer and model, depending on the position of the cuvette, the intensity of the light source, etc., and is not an absolute value.There is no problem if experiments are always performed using the same absorbance meter. In order to obtain reproducible test results including those at overseas research facilities, it was found that it was difficult to control the growth of yeast only by the OD value.
  • Sugar content (° P) is also widely used as a control index for wort fermentation, and the sugar content calculated from the specific gravity of the fermented liquor itself is called the appearance extract (appearance plateau degree). Since alcohol is generated as fermentation proceeds, the appearance extract does not accurately reflect the residual sugar concentration, but the value of the appearance extract can be easily obtained with a hydrometer or vibratory densitometer. Therefore, the effectiveness of appearance extract as a management index for proliferation was examined.
  • Table 1 shows the results of examining the relationship between the number of days of tertiary culture and the sugar content. Table 1 shows that in about 7 days of cultivation, yeast growth almost entered the stationary phase, and the amount of yeast recovered reached the maximum.
  • the yeast in the culture solution is added to the tertiary culture medium for cultivation.
  • the amount of yeast cells added at this time is also expected to affect yeast growth. Therefore, when the amount of inoculated tertiary culture was reduced, the amount of yeast growth and the amount of recovered yeast after 7 days of culturing were measured. Table 2 shows the results. As shown in Table 2, the appearance extract (° P) increased little by little and the OD 800 nm and the amount of yeast recovered (g) gradually decreased as shown in Table 2. To ensure that the stationary phase is reached near the stationary phase in 7 days of culture and the amount of yeast recovered is sufficient, the inoculum should be about 1 X 10 cells ( ⁇ 0 l X 10 10 cells). Alright, Table 2
  • HB medium (Darkose 1.5%, Maltose 6.5%, Sorbitol 2.0%, Peptone 0.2%, Yeast extract 0.4%; p H5.6] was produced and cultured in the same manner. As a result, it was found that the sugar and nitrogen consumption were close to wort.
  • the malt evaluation fermentation test was actually performed using the malt evaluation fermentation test yeast prepared using the HB medium and O medium. As a control, factory yeast conventionally used was used as it was.
  • the malt evaluation fermentation test method was carried out according to the method described in the previous report (EUROPEAN BREWERY CONVENTION MONOGRAPH XXIII EBC-SYMPOSIUM MALTING TECHNOLOGY ANDERNACH GERMANY NOVEMBER 1994, 110-136).
  • As malt to be tested as a control for everyday experiments The control malt used had good fermentability and the bad malt known to have poor fermentability was used.
  • the characteristics of yeast suitable for the malt evaluation fermentation test are that wort made with control malt consumes a good amount of sugar, and that the amount of sugar consumed and the amount of suspended yeast in a bad malt are clearly different from those of the control malt. That is, it is desirable that the difference be made.
  • Malt evaluation and fermentation tests were performed on factory malt, yeast prepared using the O medium, and yeast prepared using the HB medium using control malt and defective malt.
  • Figure 2 shows the result at 0 nm. From these results, it is clear that yeast prepared using HB medium consumes the most sugar, and wort made with poor malt consumes less sugar than control malt wort, and is clearly distinguished. I knew it could be done.
  • HB medium and O medium were found to be effective as yeast culture medium in malt evaluation fermentation tests.Therefore, several medium compositions other than HB medium and O medium were used. A modified medium was prepared, and a yeast for malt evaluation and fermentation test was prepared using the medium, and a malt evaluation and fermentation test was performed. The results are shown in Table 4.
  • the HC medium, HD medium, HE medium, and HF medium shown in Table 4 were also used in the malt evaluation fermentation test as in the HB medium. It was found to be effective as a culture medium for mother culture.
  • the use of yeast having a more stable physiological condition can be applied to the operation of factories, etc. You can obtain more reproducible test results without being affected.

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  • Life Sciences & Earth Sciences (AREA)
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Abstract

La présente invention concerne une technique de préparation destinée à un essai de fermentation en vue d'évaluer du malt à l'échelle du laboratoire, permettant ainsi d'obtenir un plus grand nombre de données d'essai reproductibles quel que soit l'état d'une plante, etc., ainsi qu'un milieu utilisé dans cette technique. On prépare une levure destinée à un essai de fermentation en vue d'évaluer du malt en cultivant une levure dans un milieu liquide contenant de 0 % à 2,5 % en masse de glucose, de 6,0 % à 9,0 % en masse de maltose, de 0 % à 2,5 % en masse de sorbitol, de 0 % à 1,0 % en masse de peptone et de 0,3 % à 1,0 % en masse d'extrait de levure entre 10° et 20° et brassé pendant 2 à 4 jours (culture primaire). Puis cette levure est cultivée dans le milieu et sa quantité est augmentée par rapport à la culture primaire, entre 10° et 20° et brassée pendant 3 à 4 jours (culture secondaire). Enfin cette culture est cultivée en quantité encore augmentée par rapport à la culture secondaire entre 8° et 9° et brassée pendant 7 à 9 jours jusqu'à ce que le degré Brix poids atteigne 3,0 °P voire moins (culture tertiaire). On laisse cette culture dans une chambre froide pendant 3 à 24 heures, puis on recueille la levure.
PCT/JP2002/001365 2001-02-19 2002-02-18 Technique de preparation de levure destinee a un essai de fermentation WO2002066673A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
DE60210749T DE60210749T2 (de) 2001-02-19 2002-02-18 Verfahren zur präparation von hefe für fermentationstest
AU2002232224A AU2002232224B2 (en) 2001-02-19 2002-02-18 Method of preparing yeast for fermentation test
JP2002566377A JP3916567B2 (ja) 2001-02-19 2002-02-18 発酵試験用酵母の調製方法
EP02712420A EP1362923B1 (fr) 2001-02-19 2002-02-18 Technique de preparation de levure destinee a un essai de fermentation
US10/468,295 US7713717B2 (en) 2001-02-19 2002-02-18 Method of preparing yeast for fermentation test
CA002438668A CA2438668A1 (fr) 2001-02-19 2002-02-18 Technique de preparation de levure destinee a un essai de fermentation

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JP2001-42051 2001-02-19
JP2001042051 2001-02-19

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US (1) US7713717B2 (fr)
EP (1) EP1362923B1 (fr)
JP (1) JP3916567B2 (fr)
AU (1) AU2002232224B2 (fr)
CA (1) CA2438668A1 (fr)
DE (1) DE60210749T2 (fr)
WO (1) WO2002066673A1 (fr)

Cited By (1)

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JP2016131512A (ja) * 2015-01-16 2016-07-25 キリン株式会社 流加培養した醸造酵母の発酵特性改善方法

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Publication number Priority date Publication date Assignee Title
JP3916567B2 (ja) * 2001-02-19 2007-05-16 麒麟麦酒株式会社 発酵試験用酵母の調製方法
KR102024197B1 (ko) * 2017-08-25 2019-09-24 대한민국 액상 형태의 주류용 효모 배양배지 및 이의 제조방법

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016131512A (ja) * 2015-01-16 2016-07-25 キリン株式会社 流加培養した醸造酵母の発酵特性改善方法

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JPWO2002066673A1 (ja) 2004-08-19
US7713717B2 (en) 2010-05-11
JP3916567B2 (ja) 2007-05-16
US20040072329A1 (en) 2004-04-15
CA2438668A1 (fr) 2002-08-29
EP1362923A1 (fr) 2003-11-19
EP1362923A4 (fr) 2004-12-29
DE60210749D1 (de) 2006-05-24
EP1362923B1 (fr) 2006-04-19
DE60210749T2 (de) 2007-03-29
AU2002232224B2 (en) 2005-08-18

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